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APPLIED AND ENVIRONMENTAL MICROBIOLOGY, May 2003, p. 2765–2772 Vol. 69, No. 5 0099-2240/03/$08.00ϩ0 DOI: 10.1128/AEM.69.5.2765–2772.2003 Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Molecular Characterization of Sulfate-Reducing in the Guaymas Basin† Ashita Dhillon,1 Andreas Teske,2‡ Jesse Dillon,3 David A. Stahl,3 and Mitchell L. Sogin1* Marine Biological Laboratory, The Josephine Bay Paul Center for Comparative Molecular Biology and Evolution,1 and Biology Department, Woods Hole Oceanographic Institution,2 Woods Hole, Massachusetts, and Department of Civil and Environmental Engineering, University of Washington, Seattle, Washington3

Received 28 October 2002/Accepted 12 February 2003

The Guaymas Basin (Gulf of California) is a hydrothermal vent site where thermal alteration of deposited planktonic and terrestrial organic matter forms petroliferous material which supports diverse sulfate-reducing bacteria. We explored the phylogenetic and functional diversity of the sulfate-reducing bacteria by character- izing PCR-amplified dissimilatory sulfite reductase (dsrAB) and 16S rRNA genes from the upper 4 cm of the Guaymas sediment. The dsrAB sequences revealed that there was a major clade closely related to the acetate- oxidizing delta-proteobacterial Desulfobacter and a clade of novel, deeply branching dsr sequences related to environmental dsr sequences from marine sediments in Aarhus Bay and Kysing Fjord (Denmark). Other dsr clones were affiliated with gram-positive thermophilic sulfate reducers (genus Desulfotomaculum) and the delta-proteobacterial Desulforhabdus amnigena and Thermodesulforhabdus norvegica. Phylogenetic anal- ysis of 16S rRNAs from the same environmental samples resulted in identification of four clones affiliated with Desulfobacterium niacini, a member of the acetate-oxidizing, nutritionally versatile genus Desulfobacterium, and one clone related to Desulfobacula toluolica and Desulfotignum balticum. Other bacterial 16S rRNA bacterial phylotypes were represented by non-sulfate reducers and uncultured lineages with unknown physiology, like OP9, OP8, as well as a group with no clear affiliation. In summary, analyses of both 16S rRNA and dsrAB clone libraries resulted in identification of members of the Desulfobacteriales in the Guaymas sediments. In addition, the dsrAB sequencing approach revealed a novel group of sulfate-reducing prokaryotes that could not be identified by 16S rRNA sequencing.

The Guaymas Basin is one of the series of deep semiclosed Basin sediments have adapted to the petroleum hydrocarbon- basins in the Gulf of California (5) where tectonic activity of rich environment, e.g., the anaerobic sulfate-reducing bacteria the site generates a high heat flow (19). The highly productive that oxidize alkanes, aromatic compounds, and fatty acids (30, surface waters and terrigenous input from Baja California and 34) and aerobic aromatic hydrocarbon-degrading bacteria with the Mexican mainland lead to rapid sediment accumulation a preference for aromatic carboxylic acids (11). Thus, the mi- and have created sediment layers up to 400 m deep (36). crobial communities in the Guaymas Basin may depend more Hydrothermal fluids are discharged both through chimneys at on the utilization of hydrocarbon constituents derived primar- 270 to 325°C and through the porous sediment (21). Compared ily from photosynthetic biomass than on chemoautotrophic to other known hydrothermal vent sites, the Guaymas Basin is carbon assimilation with sulfide oxidation as an energy source. unique in that the organic matter in the accumulating sediment The sulfate-reducing bacteria (SRB) are a large and ex- is pyrolized under high-temperature conditions to petroleum- tremely diverse physiological group of anaerobic microorgan- like products, such as gasoline-range aliphatic and aromatic isms capable of degrading a wide range of organic substrates, hydrocarbons and residual polar asphaltic material (2, 35, 36). including petroleum-based products like alkanes (34, 37), tol- Petroleum is thought to undergo microbial degradation at uene (4, 32), benzene (30), and polyaromatic hydrocarbons precipitated mineral mound surfaces or unconsolidated sedi- (10, 51). The strictly anaerobic life style and slow growth rate ments, as well as the sediment-water interface (2, 35). In ad- of SRBs make it very difficult to isolate and identify represen- dition to petroleum hydrocarbons, the chemical milieu of the tatives that grow on hydrocarbons. A culture-independent al- Guaymas vents includes short-chain organic acids and ammo- ternative approach based on the 16S rRNA gene sequence has nia, which are released via pyrolysis of organic material in the been used to characterize bacterial communities, including sediments (23, 44). Many bacterial populations in Guaymas novel biodegraders (1, 29, 37, 51). Sequence analyses of PCR- amplified 16S rRNA coding regions provide the most general framework for studies of natural microbial diversity and abun- * Corresponding author. Mailing address: Marine Biological Labo- ratory, Josephine Bay Paul Center for Comparative Molecular Biology dance (8, 15, 29, 33). However, 16S rRNA-based analysis does and Evolution, NASA Astrobiology Institute, 7 MBL St., Woods Hole, not provide an unambiguous link to the physiology or meta- MA 02543. Phone: (508) 289-7246. Fax: (508) 457-4727. E-mail: sogin bolic capabilities of a bacterium, particularly in newly discov- @mbl.edu. ered phylogenetic lineages without cultured isolates and † This is Woods Hole Oceanographic Institution publication no. 10905. known phenotypes (6, 8, 15). Hence, the functional gene ap- ‡ Present address: Dept. of Marine Sciences, University of North proach has been used to identify bacteria responsible for bio- Carolina, Chapel Hill, Chapel Hill, N.C. geochemical processes in the environment (7, 16, 25).

2765 2766 DHILLON ET AL. APPL.ENVIRON.MICROBIOL.

FIG. 1. (A) Phylogenetic tree based on the translated amino acid sequences of PCR-amplified dsrAB genes from sulfate-reducing prokaryotes. Sequences were retrieved from Guaymas core B sediment layers (1 to 4 cm). The tree was constructed by using the maximum-likelihood method in PUZZLE. The bootstrap analysis was done by using puzzleboot and 100 replicates. Bootstrap values are shown for branches with more than 50% bootstrap support. (B) Phylogenetic tree based on the translated, partial amino acid sequences of dsrAB genes from sulfate-reducing prokaryotes. Sequences were retrieved from Guaymas core B sediment layers (1 to 4 cm). The tree was constructed by using the maximum-likelihood method in PUZZLE. The bootstrap analysis was done by using puzzleboot and 100 replicates. Bootstrap values are shown for branches with more than 50% bootstrap support. Group I, group II, and group III sequence clusters were defined as described in reference 43; group IV was based on this data set. VOL. 69, 2003 SRB IN THE GUAYMAS BASIN 2767

FIG. 1—Continued. FIG. 2. Phylogenetic tree based on 16S rRNA sequences of bacterial clones from Guaymas core B sediment layers (1 to 4 cm). The tree was constructed with PAUP*. Bootstrap values based on 500 replicates each (for distance and parsimony) are shown for branches with more than 50% bootstrap support.

2768 VOL. 69, 2003 SRB IN THE GUAYMAS BASIN 2769

A key enzyme of dissimilatory sulfate reduction, dissimila- Phylogenetic analysis. The dsrAB sequences were translated by using Mac- tory sulfite reductase (EC 1.8.99.3), catalyzes the reduction of Clade (http://macclade.org) and were aligned by using ClustalX (42). We used the quartet puzzling algorithm in PUZZLE, version 5.0, to estimate a tree with sulfite to sulfide, an essential step in the anaerobic sulfate maximum-likelihood branch lengths. A bootstrap analysis was done by using respiration pathway. The key gene coding for this essential puzzleboot v 1.03 script. For searches under distance criteria we used 100 rep- enzyme has been found in all SRBs that have been tested so far licates per data set. The analysis included the full-length translated region of the (17, 45). The 1.9-kb DNA fragment encoding most of the ␣ and dsrAB genes, excluding the region between the stop codons. In the case of the ␤ subunits of dissimilatory sulfite reductase can be amplified by partial dsrAB tree, partial regions corresponding the Aarhus Bay and Kysing Fjord (Denmark) forward and reverse sequences were used in the analysis. The PCR from all recognized lineages of SRBs (17, 45). Compar- 16S rRNA sequence alignment for phylogenetic inference was minimized by ative amino acid sequence analysis of the dissimilatory sulfite using a mask in ARB (1,296 positions were used for analysis). The dendrogram reductase genes (dsrAB) has recently been used to investigate was constructed with the ARB database evolutionary distance (neighbor-joining the evolutionary history of anaerobic sulfate (sulfite) respira- algorithm with likelihood settings). Distances were calculated by using the best- fit model TrNϩIϩG, as selected by Model Test, version 3.06 (31). The robust- tion (13, 17, 45) and the environmental diversity of uncultured ness of inferred topologies was tested by bootstrap resampling with minimum SRB populations (7, 16, 25, 43). In this study we investigated evolution and parsimony methods implemented in PAUP*, version 4.08b (39), by dsr diversity in the Guaymas Basin using full-length sequences using the same distance model. The 16S rRNA analyses were checked for of PCR-amplified dsrAB gene fragments. This is the first report chimeras by using the CHIMERA-CHECK online analysis program of the of PCR-amplified dsrAB sequences from environmental DNA RDP-II database (22). Three chimeras that were identified were removed from the phylogenetic analysis. For the dsrAB sequences, complete and partial dsrAB that include nearly full-length alpha- and beta-subunit se- gene trees gave the same phylogenetic structure, ruling out the possibility of quences. chimeras. The amino acid alignment was also checked for ambiguities visually. Blast searches were done for complete dsrAB fragment nucleotide sequences; the deeply branching group did not show any statistically significant similarity to MATERIALS AND METHODS other dsrAB sequences in the database. Sampling sites and characteristics. Sediment cores were retrieved during Nucleotide sequence accession numbers. The Guaymas Basin 16S rRNA and dives with the research submersible Alvin (Woods Hole Oceanographic Institu- dsrAB gene sequences have been deposited in the GenBank database under the tion) on a cruise to the Guaymas Basin in April 1998. Core B (Alvin dive 3205; following accession numbers: 16S rRNA, AY197374 to AY197384, AY197386, 28 April 1998; diameter, 6 in.; length, 25.3 cm) was obtained from the Everest AY197388 to AY197403, AY197405 to AY197424; and dsrAB, AY197425 to Mound area in the Southern Guaymas vent field (27°00.888ЈN, 111°24.734ЈW). AY197459. The designations of various clones obtained from core B from the Guaymas Basin begin with B, while the designations of clones obtained from core A (dive 3203; Everest Mound; 27°00.762NЈ, 111°24.656WЈ) and core C (dive 3207; Or- RESULTS pheus site; 27°00.435NЈ, 111°24.612W) begin with A and C, respectively, as described in a previous study (40). Core B had no Beggiatoa mat, and the upper Diversity of dsrAB genes. Five different phylogenetic groups 6 cm consisted of a black layer of liquid petroleum mud. Gas bubbles appeared of dsrAB genes were found in the Guaymas sediment (Fig. 1A). in the sediment at a depth of 7 cm. The in situ temperature profile of the sediment obtained by using Alvin’s thermoprobe was determined prior to coring. Nearly 34% of the sequences were closely related to dsrAB The uppermost 5 cm of the core had a temperature range of 3 to 16°C, while the genes of the gram-negative delta- Desulfobacter temperature range for the segment from 5 to 23 cm was 16 to 140°C. The cores latus and Desulfobacter vibrioformis, which are members of the were brought to the surface within6hofsampling and were sliced at 1-cm acetate-oxidizing genus Desulfobacter (Fig. 1A). A second intervals within 6 h after retrieval. Multiple 2-ml portions of the sediments were dsrAB cluster that accounted for 57% of the sequences ampli- taken from each 1-cm layer and immediately frozen at Ϫ80°C for nucleic acid extraction. fied from Guaymas sediments branched deeply in the dsrAB Nucleic acid extraction, 16S rRNA and dsr gene amplification, cloning, and tree and was phylogenetically distinct from sequences of cul- sequencing. Total genomic DNA was extracted from 30 mg of sediment from tured strains (Fig. 1A). This cluster contained two distinct each layer by using a MOBIO cloning kit (MO BIO Labs Inc., Solana Beach, lineages; two novel dsrAB sequences (B04P026 and B04P037) Calif.). Bacterial oligonucleotides 8F and 1492R (40) were used to prime the 16S rRNA PCRs. We employed primers dsr1F and dsr4R (45) to amplify the dsr formed a sister group to a tightly clustered assemblage of 18 genes from environmental samples. Each PCR mixture (final volume, 50 ␮l) dsrAB sequences whose phylogenetic affinity was uncertain contained 5 ␮l of each primer solution (10 pmol/␮l), 1 ␮l of DNA, 5 ␮lof10ϫ (Fig. 1A). Only small numbers of other dsrAB sequences were PCR buffer, 1.25 ␮lof10ϫ bovine serum albumin (10 mg/ml), 5 ␮lofa10ϫ found. A single dsrAB sequence (B01P021) from the Guaymas deoxynucleoside triphosphate mixture (2 mM dATP, 2 mM dCTP, 2 mM dGTP, sediments formed a monophyletic lineage with Thermodesul- 2 mM dTTP), and 2.5 ␮lofTaq DNA (2 U/␮l). Amplification was carried out with an Eppendorf Mastercycler gradient PCR machine. After initial denatur- forhabdus norvegica and Desulforhabdus amnigena. Two dsrAB ation for 1 min at 94°C, 30 cycles were performed, with each cycle consisting of sequences grouped with a dsr clade of thermophilic, gram- 1 min at 94°C, 1 min at 54°C, and 3 min at 72°C. The reaction was completed by positive SRB, represented by Desulfotomaculum thermocister- a final extension step at 72°C for 1 min. PCR products were purified on an 0.8% num, Desulfotomaculum thermoacetoxidans, and Desulfo- agarose gel and then poly(A) tailed and cloned by using a TOPOXL PCR cloning kit before they were transformed into Escherichia coli by using the manufactur- tomaculum thermosapovorans. er’s protocols (Invitrogen, San Diego, Calif.). Comparisons with partial dsrAB sequences from environ- The dsrAB sequences were determined with an ABI 3700 by using the M13 mental samples that included the alpha and beta subunits pro- universal primers SP010 and SP030 to initiate DNA synthesis in cycle sequencing vided additional resolution while confirming the mutually ex- reactions. The 16S rRNA sequences were determined with a LICOR 4200 clusive, well-supported major branches of the dsrAB trees sequencer by using the same sequencing primers. The 16S rRNA sequences were assembled by using Phrap (12) and were aligned with sequences from the Gen- based on full-length dsrAB fragments (Fig. 1B). The deeply Bank database by using the ARB software (www.arb-home.de) fast aligner util- branching Guaymas sequences lacking cultured relatives were ity. To obtain full-length dsr sequences, the internal degenerate sequencing related but were not identical to sequences from coastal ma- Ј Ј Ј primers 1FI (5 -CAGGAYGARCTKCACCG-3 ) and 1R1 (5 -CCCTGGGTRT rine sediments, including clones KYF 324 and KYF 135 from GRAYRAT-3Ј) were designed on the basis of sequence conservation around positions 500 and 1500 in the dsrAB amplicons. The dsrAB sequences were Kysing Fjord and related dsrAB lineages from Aarhus Bay, assembled by using the software package Sequencher (http://www.geneco- Denmark (43) (Fig. 1B). None of the dsrAB sequences from des.com). the Desulfobacter-related delta-proteobacterial group from the 2770 DHILLON ET AL. APPL.ENVIRON.MICROBIOL.

Guaymas Basin or from cultured isolates were related to De- acetate- and short-chain fatty acid-rich surface layers of the sulfobacter-related environmental sequences (group I) isolated Guaymas sediments (21). from Kysing Fjord and Aarhus Bay (Fig. 1B) (43). Two nearly identical dsrAB sequences (B04P001 and Bacterial 16S rRNA gene sequences. The bacterial clone B04P011) grouped with dsrAB genes of thermophilic represen- libraries from the Guaymas sediments were very diverse and tatives of the gram-positive genus Desulfotomaculum, including included relatives of numerous cultured and uncultured lin- Desulfotomaculum thermocisternum, Desulfotomaculum ther- eages in the Proteobacteria, as well as numerous other bacterial moacetoxidans, and Desulfotomaculum thermosapovorans. The phyla (Fig. 2), and some were related to clones from a previous phylogenetic positions of thermophilic Desulfotomaculum spe- study (40). Most 16S rRNA gene sequences of delta-pro- cies in dsrAB and rRNA gene trees are discordant. Alternative teobacterial SRB (B02R021, B04R008, B01R024, B01R011, explanations for this include possible lateral gene transfer (17) B01R004) and the previously sequenced Guaymas clone or ancestral gene duplication followed by differential loss in the CSB008 (40) clustered with Desulfobacterium niacini and Des- genus Desulfotomaculum. The nutritional demands of these ulfobacterium autotrophicum (Ͼ90% bootstrap support). Mem- sulfate reducers match the abundance of acetate and short- ber of the acetate-oxidizing, nutritionally versatile genus Des- chain fatty acids in the Guaymas sediments. Desulfotomaculum ulfobacterium also degrade short-chain fatty acids, ethanol, species degrade a wide spectrum of substrates, including ace- lactate, and tricarboxylic acid (TCA) cycle intermediates (48, tate, short-chain fatty acids, ethanol, and lactate (47), while in 49). One Guaymas clone (B02R015) was related to the acetate the absence of sulfate they can grow acetogenically on benzo- and aromatic hydrocarbon oxidizers Desulfobacula toluolica ate, propionate, butyrate, and ethanol (41). (32) and Desulfobacterium phenolicum (48). In brief, the 16S The dsrAB clone related to Desulforhabdus amnigena and T. rRNA sequences were most closely related to the sequences of norvegica (Fig. 1A) indicated that SRB that were terminal cultured SRB that oxidize acetate and a wide range of sub- oxidizers of acetate with the potential for aromatic hydrocar- strates completely to CO2. The most common bacterial phylo- bon degradation were present. Desulforhabdus amnigena has types in core B with significant numbers of clones were mem- been isolated from anaerobic granular sludge and uses acetate bers of the epsilon subclass of the Proteobacteria, the as a sole carbon and energy source (28). It does not metabolize Cytophaga-Flavobacterium-Bacteroides phylum, the uncultured benzoate or other aromatic compounds, but its close relative, bacterial group associated with methane- and hydrocarbon- the sulfate-reducing strain PROTL1, is known to be a toluene rich sediments, and an uncultured lineage with no clear phy- degrader (4). The thermophilic gram-negative species T. nor- logenetic affiliation (Fig. 2). Other phylogenetic lineages that vegica oxidizes acetate, lactate, TCA cycle intermediates, and contained only one or two Guaymas clones included the short-chain fatty acids; it was isolated from hot (70 to 75°C) oil gamma subclass of the Proteobacteria, the Spirochaetes, the field water samples, an environmental setting similar to the Verrucomicrobia, the Holophaga-Acidobacterium lineage, the Guaymas Basin (3). At present, it is not possible to identify the green nonsulfur bacterial phylum, the Firmicutes, and the un- SRB carrying the new clade of dsrAB sequences that are re- cultured WS3 and WS6 lineages obtained previously from a jet lated only to environmental dsrAB sequences from Kysing fuel-contaminated aquifer (8). Also found were the uncultured Fjord; no cultured isolate is available that can connect dsrAB OP8 candidate subdivision from deep-sea sediments and geo- and 16S rRNA phylotypes. Although in a previous study the thermal springs (15), sequences related to clone SJA43 from workers demonstrated that there are anaerobic methane-oxi- an anaerobic trichlorobenzene-degrading bioreactor (50), and dizing microbial communities in Guaymas sediments (40), the a sequence related to SAR406. dsrAB data set does not allow unambiguous identification of syntrophic SRB or their substrates that are involved in this process (14, 38). The sulfate-reducing members of anaerobic DISCUSSION methanotrophic consortia in Guaymas sediments so far have Diversity of dsrAB genes. The dsrAB sequences within the not been identified by fluorescence in situ hybridization. 16S Desulfobacteriales, close to and Desul- rRNA probes for members of the genus Desulfosarcina, the fobacter vibrioformis, could represent true Desulfobacter species common sulfate-reducing syntrophs in methane-oxidizing con- or dsrAB genes of the gram-negative, delta-proteobacterial sortia, have not hybridized with the bacterial cells in consortia species Desulfobacula toluolica (32; M. Wagner, unpublished from Guaymas Basin and several other locations (18, 27). Des- results). The latter possibility is supported by the sequence of ulfosarcina sequences were not found in this survey and were a 16S rRNA clone related to Desulfobacula toluolica in the 16S not found in previous 16S rRNA-based studies (40). The dsrAB rRNA library (B02R015). Furthermore, the current dsrAB da- sequences without any cultured relatives that are predominant tabase does not include representatives of the closely related in this data set were most closely related to dsrAB clones from genus Desulfobacterium (17, 45), a genus of nutritionally ver- sulfate-reducing sediment layers and not to phylogenetically satile acetate oxidizers that is well represented in the 16S distinct dsrAB clones from the methane-oxidizing zone (43). rRNA clone library of the Guaymas Basin (Fig. 2). Therefore, Bacterial diversity in the Guaymas sediments. Most of the the Desulfobacter-related dsrAB sequences retrieved from the rich bacterial diversity in Guaymas core B was affiliated with Guaymas sediments could be derived from SRB belonging to sequences isolated from hydrocarbon-rich environments, cold the genus Desulfobacterium, whose 16S rRNA genes were seep environments, or hydrocarbon-rich enrichments (20, 26, found frequently in the Guaymas samples. Acetate-oxidizing 30). The diverse proteobacterial and nonproteobacterial lin- and short-chain fatty acid-degrading, mesophilic SRB of the eages in this study represent a mixture of phylotypes similar to genera Desulfobacter, Desulfobacula, and Desulfobacterium that found in a previous study (40), related to presumably should find favorable growth conditions in the relatively cool, sulfur-oxidizing lineages (gamma and epsilon subclasses of the VOL. 69, 2003 SRB IN THE GUAYMAS BASIN 2771

Proteobacteria) and to phylotypes frequently found in geother- thermophilic Desulfotomaculum species and of T. norvegica;an mal springs, hydrocarbon-rich cold seeps, and anthropogenic additional peak occurred between 80 and 90°C, which matched oil spills (8, 15). the temperature optimum of archaeal sulfate reducers of the The 16S rRNA clones of SRB belonging to the phylotype genus Archaeoglobus. With increasing sediment depth, the Desulfobacteriales found in this study represent a group of temperature profile of sulfate-reducing activity moved towards organisms capable of complete oxidation of acetate and other the range of Archaeoglobus, and the overall sulfate reduction electron donors, such as short-chain fatty acids, TCA cycle rates decreased (46). Apparently, the PCR-based survey did intermediates, and aromatic compounds. These substrates are not detect Archaeoglobus-related dsrAB genes, although the end products of hydrolysis and fermentation of complex poly- activity of these hyperthermophilic sulfate reducers could be meric compounds by the diverse, heterotrophic microbial pop- measured. These results emphasize the need for quantitative ulations in the Guaymas Basin (11, 40) and, in addition, the molecular approaches in microbial community analysis that products of thermogenic petroleum generation in the Guay- circumvent PCR and cloning biases. mas sediments (35, 36). Candidates for novel SRB are not necessarily limited to the ACKNOWLEDGMENTS classical delta-proteobacterial SRB. The highly diverse mem- This study was supported by the NASA Astrobiology Institute (grant bers of the uncultured phylum candidate divisions OP8 and CAN NCC2-1054) and by the G. Unger Vetlesen Foundation. Sam- phylogenetic groups with no clear affiliation could include pre- pling at the Guaymas Basin was made possible by NSF Life in Extreme viously unrecognized SRB that fill the niche. For example, Environments grant OCE 9714195 to A.T. members of the lineage associated with hydrocarbon- and Molecular analyses were carried out at the W. M. Keck Ecological and Evolutionary Genetics Facility in the Josephine Bay Paul Center methane-rich sediments were found repeatedly in sulfate-re- for Comparative Molecular Biology and Evolution at the Marine Bi- ducing enrichments from Guaymas sediment (30) and in ological Laboratory. freshly harvested Guaymas sediment characterized by high sul- fate reduction rates (40). Although their environmental distri- REFERENCES 1. Amann, R. I., W. Ludwig, and K. H. Schleifer. 1995. 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